Preliminary Study On The Immunomodulatory Mechanism Of Different CpG ODNs In Aquatic Crustaceans

The frequent outbreak of diseases in aquaculture animals produces harmful effect on aquaculture in China.The development of environmentally friendly immune substance has received great attention in aquaculture,among which,CpG ODNs(Oligodeoxynucleotides)have received extensive attention due to their great antibacterial and antiviral effects.In the present study,the recognition effect and the immune responses of different CpG ODNs in Litopenaeus vannamei and Eriocheir sinensis were explored by means of molecular biology and cell biology techniques,and their antiviral effect and related mechanism were further investigated,providing a theoretical basis for the application of CpG ODNs in aquaculture.Four types of CpG ODNs(CpG-A,CpG-B,CpG-C and CpG-P)were selected to stimulate shrimps,and then shrimps were challenged with WSSV to detect the virus copy numbers in their gill tissue.The virus copy numbers after five days post injection in CpG-A,CpG-B,CpG-C and CpG-P group were 18.19%,48.82%,57.75% and 47.65% of that in PBS group,respectively.It showed that four types of Cp G ODNs could inhibit WSSV copy numbers in different degree(CpG-A > CpG-P > CpG-B > CpG-C)and CpG-A played an important role in the process of antiviral immune response.The immunological recognition activities of LvTolls(Lv Toll1,LvToll2,LvToll3)to different CpG ODNs and the mRNA expression levels of LvTolls after CpG ODNs stimulation were investigated.The mRNA expression level of three Lv Tolls in hepatopancreas increased significantly at 24 h post CpG-A stimulation.CpG-P could also significantly enhance the expression level of LvTolls,but the increase amplitude in CpG-B and CpG-C groups were relatively less.In addition,the mRNA expression level of LvToll1 was significantly higher than that of Lv Toll2 and LvToll3 in CpG-A group.In vitro experiment,CpG-A showed the strongest binding ability with three LvToll-ECDs.The binding ability of LvToll1-ECD with CpG-A was also significantly stronger than that of LvToll2-ECD and LvToll3-ECD with CpG-A,It indicated that CpG-A was an important type of CpG ODNs that could activate the Toll pathway in shrimp,and LvToll1 may be the main receptor of CpG-A in Litopenaeus vannamei.The downstream immune responses induced by the four different types of CpG ODNs were explored.CpG-A and CpG-B could enhance phagocytosis and ROS levels of hemocytes,inducing cellular immune response.Relevant indicators in the IRF-Vago-JAK/STAT pathway were tested in shrimp.The expression levels of Lv IRF,LvVago4,LvVago5 and Lv STAT were significantly up-regulated after CpG-A treatment,and the IFN content in plasma also increased,showing that CpG-A could activate the IFN system-like antiviral system,playing an important role in shrimp antiviral immunity.In addition,CpG-B could improve the content of TNF in plasma;CpG-C could increase the plasma levels of IL;CpG-A and CpG-P could improve the plasma level of IFN,revealed that different types of CpG ODNs showed different effects in shrimps,which were similar to their roles in mammals.CpG ODNs related immunity indicators were detected after RNA interference of EsMyD88,to explore the mechanism of the immune responses activated by CpG ODNs in crustaceans.Results showed that mixed CpG ODNs could improve the mRNA expression levels of several immune related genes(EsProPO,CrusEs2,EsCAM,EsLITAF and EsmtMnSOD).But compared with the CpG ODNs treatment group,the expression levels of these genes in co-treatment of EsMyD88 interference and CpG ODNs group were significantly reduced.Moreover,the increase of IFN,IL and CAT(catalase)activity in plasma caused by CpG ODNs could also be reduced by EsMyD88 interference.The above results indicated that part of the immune functions induced by CpG ODNs in Eriocheir sinensis were mediated by EsMyD88.In conclusion,as a novel immunostimulant,CpG ODNs could trigger the immune responses and induce great antiviral effect in crustaceans through the activation of Toll pathway.And different types of CpG ODNs showed diverse effects,providing a theoretical basis for their application in aquaculture.